Decalcification Solution with Preservation of RNA

ABSTRACT

The present invention is directed to methods of decalcification and tissue sample preparation that allows for the reproducible quantitative analysis of gene expression in hard tissue samples like bone, mineralizing cartilage and tendon, dentin, cementum and/or enamel that are too hard to section effectively using conventional means.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 61/667,557 entitled “Decalcification Solution withPreservation of RNA,” filed Jul. 3, 2012, and incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology and morespecifically the preparation of bone or other hard tissue samples forgene expression analysis and/or other analyses where RNA preservation isimportant.

BACKGROUND OF THE INVENTION

In scientific research studies in the general area of cell biology,investigations of both soft and hard (mineralized) tissues arecommonplace. Examination and analysis of hard tissues such as bones andteeth, the shells of mollusks and other examples of vertebrate,invertebrate, plant, bacterial and other organisms often encounterdifficulties since these tissues in many instances must be speciallytreated to remove their constituent mineral. For investigations todetermine nucleic acids, genes, and proteins of both soft and hardtissue cells, a frequent method of processing involves cutting the cellsinto very thin sections. Again, the demineralization of the hard tissuesis required for proper and consistent sectioning.

Decalcification can be accomplished by utilizing any of severaldifferent methodologies, including application of chelating reagents,acids or even microwave radiation. There are many products currently onthe market and available for demineralization, intended to assist inhard tissue treatment for routine clinical and basic science research inthe field of protein analysis.

For aspects of cell biology concerning nucleic acid, and particularlyribonucleic acid (RNA), research and gene studies, additional techniquesmust be carefully considered. Because gene expression can change withinseconds of cell processing, tissues must be handled in a way that doesnot change or degrade (destroy) the cellular RNA. In the cases of human,animal or plant tissues, RNA analysis had generally required that thesamples be snap-frozen in liquid nitrogen at the immediate point oftheir retrieval upon death or surgical removal. Snap-frozen RNA hasproven to be of the highest quality and other methods are usuallycompared to this standard.

More recently, however, RNA preservation solutions like RNAlater®Solution have been developed to stabilize RNA in animal, human or othertissues without the need for snap freezing. It has been found that,samples stored in RNAlater® Solution were of a quality comparable tosamples processed and stored following snap-freezing. This was asignificant advance in nucleic acid preservation, since it was oftenimpossible, especially away from the laboratory and out in the field, tohave access to or carry the liquid nitrogen necessary for snap freezing.

The importance of RNA preservation has continued to grow with thedevelopment of more and more sophisticated genetic analyses usingvarious types of RNA. In particular, researchers and medicalprofessionals have been utilizing gene expression analysis to bothdiagnose and treat various maladies and for basic scientific research.While each cell in an organism will contain the same genomic DNA andaccordingly the same “genes,” only a small fraction of the genes in anyparticular cell or cell type is ever used at any one time. When aparticular gene is activated, the genetic information necessary tocreate the prescribed protein is transcribed to the ribonucleic acid(RNA) that will be used to make the desired protein.

By identifying and quantifying the RNA in a sample, gene expressionanalysis makes it possible to determine both what genes are beingexpressed and, often more importantly, when, where, and in whatconcentration those genes are being expressed. That is, if the locationfrom which the RNA was recovered can be accurately determined, then theparticular cells or cell types that are responsible for the geneexpression can likewise be determined. The location of the cells or celltypes studied, and, therefore the source of the RNA recovered from thesample, is ordinarily a matter of careful sectioning of the sample to betested. From the sample section, specific cells, cell types etc. can beselected for analysis using techniques such as manual or laser capturemicrodissection. Two increasingly common types of gene expressionanalyses are In-Situ Hybridization (ISH) and laser capturemicrodissection (LCM).

ISH analysis of RNA requires sections to be cut from chemically fixedtissue samples and then molecular probes are used to label and identifygenes of interest in these sections. RNA may be visualized by thelabeling in particular areas of the sections and analyzed qualitativelyin a temporospatial manner. The detection of labeled genes in hardtissues again requires demineralization to achieve optimal results.

LCM methodology is unique compared to other procedures for gene (andprotein) identification in that specific cells of interest, identifiedby viewing them under a microscope within a population of cells, may beprecisely removed from a tissue section using a laser beam. The isolatedand so-called captured cells may then be analyzed to address a multitudeof questions dealing with gender, disease, drug and other effects on aspecimen. RNA obtained following LCM capture of one or several cells insections may subsequently be assessed quantitatively using reversetranscription-quantitative polymerase chain reaction (RT-qPCR) analysis.Resulting data offer insight into biochemical reactions and pathways ina more direct manner than that of ISH. LCM is a microscopic techniquerequiring tissues to be sectioned onto particular slides of either glassor a thin polymer membrane. Hard tissue study by LCM normally requiresdemineralization of the sample.

A common approach to analyze specific cell types, individually or asgroups of cells spatially, is cryosectioning. Samples are snap-frozenand embedded in a tissue freezing medium available commercially. Thefrozen tissue or biopsy is sectioned into 4-20 μm slices in a cryostatinstrument and kept frozen to preserve the RNA until isolation andanalysis. This routine procedure for analysis is easily accomplished ontissues that are by nature soft, i.e., kidney or liver. This approachdoes make it possible to determine with some precision what cells andcell types are being analyzed.

Analysis of hard tissues like bone, mineralizing cartilage and tendon,dentin, cementum, and enamel, as well as invertebrate shells and othertissues which are too hard to cut or section effectively usingconventional means, such as a cryostat has been found, however, to be amajor problem. There have developed a variety of approaches to preparingthese types of hard tissues for gene expression analysis. One commonapproach is to snap-freeze and then grind the hard tissue into a powder,and extract the RNA for analysis. While this approach does produce RNAfor gene expression analysis relatively quickly without significantdegradation, the gene data obtained using this method relate to all ofthe cells present in the sample. Because of this, it is not possible todetermine what specific phenotype or lineage in population of cells/celltypes present in the typically mixed cell groupings are producing theRNA obtained.

Another approach is to decalcify the hard tissue sample with acids orchelating agents, thereby softening it so it can be cryosectioned andanalyzed. The two most common groups of decalcifying agents known in theart are chelating agents and acids. The acids may be further dividedinto weak organic (picric, acetic and formic acid) and strong inorganicacids (nitric and hydrochloric acid). The acids dissolve hydroxyapatitemineral with release of calcium ions while chelating agents take up orcapture the calcium ions within their structure. The most frequentlyclinically used chelating agent is ethylenediaminetetraacetic acid(EDTA).

Unfortunately, however, RNA is relatively fragile and tends to breakdown rapidly after the tissue sample is taken and known decalcificationagents have been found to degrade the RNA in the sample. Acids used fordecalcification in other procedures are very harsh, and with chelatingagents such as EDTA or disodium-EDTA, the decalcification process cantake as long as four to six weeks. The RNA recovered using this processis often degraded to the point that it can not be quantitated in anyreliable manner. While increasing the concentration of thedecalcification agent has been found to speed decalcification, it alsoincreases the rate of degradation of the RNA in the sample.

Accordingly, there is a need in the art for a method for the rapiddecalcification of hard tissues like bone, mineralizing cartilage andtendon, dentin, cementum and other tissues that are too hard to sectioneffectively using conventional means, which preserves the ability todetermine the site of gene expression without significantly degradingthe RNA recovered for analysis.

SUMMARY OF THE INVENTION

The present invention is directed to a method of decalcification thatallows the reproducible quantitative analysis of gene expression forhard tissue samples like bone, mineralizing cartilage and tendon,dentin, cementum and other tissues that are too hard to sectioneffectively using conventional means.

In one aspect, the present invention is directed to a method ofpreparing a decalcification agent solution for the decalcification ofhard tissues like bone, mineralizing cartilage and tendon, dentin,cementum and enamel without degrading the ribonucleic acids (RNA)contained therein, comprising the steps of: providing an RNApreservation solution; adding a decalcification agent to said RNApreservation solution; wherein said decalcification agent furthercomprises tetrasodium-ethylenediaminetetra-acetic acid(tetrasodium-EDTA) or trisodium-ethylenediaminetetra-acetic acid(trisodium-EDTA); adjusting the pH of the mixture to a pH of from about8 to about 10 and stirring until the decalcification agent issubstantially dissolved; readjusting the pH of the mixture to a pH offrom about 7.2 to about 7.7; and sterilizing and collecting the mixturein a sterilized container.

In some embodiments of the present invention, the decalcification agentis tetrasodium-ethylenediaminetetra-acetic acid (tetrasodium-EDTA). Insome embodiments, the method of the present invention may include any ofthe embodiments described above wherein the mixture is stirred for fromabout 30 minutes to about 60 minutes. In some embodiments, the method ofthe present invention may include any of the embodiments described abovewherein the pH of the mixture is adjusted to a pH of about 7.4.

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein the mixture is sterilized byfiltration. In some embodiments, the method of the present invention mayinclude any of the embodiments described above wherein the mixture issterilized using a 0.2 μm filter.

In another aspect, the present invention is directed to a method ofdecalcification of hard tissues like bone, mineralizing cartilage andtendon, dentin, cementum, enamel and other examples for sectioningwithout degrading the ribonucleic acids (RNA) contained therein,comprising the steps of: obtaining a hard tissue sample and placing itin an RNA preservation solution; removing said hard tissue sample fromthe RNA preservation solution and placing it in a liquid decalcificationmedium comprising an RNA preservation solution and a decalcificationagent, wherein said decalcification agent is selected from the groupconsisting of tetrasodium-EDTA or trisodium-EDTA wherein saiddecalcification agent begins to remove calcium from mineral deposits insaid hard tissue sample; stirring the mixture said until said hardtissue sample softens for sectioning; and rinsing the softened hardtissue sample in an RNA preservation solution to remove any remainingdecalcification agent.

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein said hard tissue sample is atissue sample selected from the group consisting of bone, mineralizingcartilage and tendon, dentin, cementum, enamel and others. In someembodiments, the method of the present invention may include any of theembodiments described above wherein the size of said hard tissue sampledoes not exceed 0.5 cm, on one side.

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein said hard tissue sample isplaced in said RNA preservation solution for a period of from about 18hours to about 24 hours at a temperature of from about 3° C. to about 4°C. In some embodiments, the method of the present invention may includeany of the embodiments described above wherein said hard tissue sampleis placed in said RNA preservation solution for about 1 day at atemperature of about 4° C. In some embodiments, the method of thepresent invention may include any of the embodiments described abovewherein said decalcification agent istetrasodium-ethylenediaminetetra-acetic acid (tetrasodium-EDTA).

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein the ratio of the volume ofsaid decalcification agent to the weight of said hard tissue sample isfrom about 10:1 to about 20:1. In some embodiments, the method of thepresent invention may include any of the embodiments described abovewherein the mixture is stirred for from about 3 days to about 7 days. Insome embodiments, the method of the present invention may include any ofthe embodiments described above wherein the liquid decalcificationmedium is changed from every about 36 to every about 48 hours. In someembodiments, the method of the present invention may include any of theembodiments described above wherein the decalcification agent is removedfrom said tissue sample by rinsing said tissue sample in an RNApreservation solution for a period of time from about 12 hours to about24 hours. In some embodiments, the method of the present invention mayinclude any of the embodiments described above further comprising thestep of: rinsing the tissue sample in RNase-free water to remove anyremaining RNA preservation solution.

In another aspect, the present invention is directed to a method ofpreparing hard tissues like bone, mineralizing cartilage and tendon,dentin, cementum, enamel and other examples for gene expression analysiscomprising the steps of: obtaining a hard tissue sample; trimming saidhard tissue sample to a minimum size needed for gene expressionanalysis, wherein said tissue sample has at least one edge that is lessthan 0.5 cm in length; placing said hard tissue sample in an RNApreservation solution; removing said hard tissue sample from the RNApreservation solution and placing it in a liquid decalcification mediumcomprising a decalcification agent selected from the group consisting oftetrasodium-EDTA or trisodium-EDTA and an RNA preservation solution,wherein said decalcification agent begins to remove calcium from mineraldeposits in said hard tissue sample, causing said hard tissue sample tosoften; stirring the mixture until enough of the calcium has beenremoved from the mineral deposits in said hard tissue sample to causethe tissue sample to soften enough to permit sectioning of said tissuesample; rinsing the softened hard tissue sample in RNA preservationsolution and then RNase-free water; soaking the softened hard tissuesample in cryoembedding medium for from about 45 minutes to about 60minutes; freezing the infiltrated sample and cryosectioning the softenedhard tissue sample.

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein said hard tissue sample is atissue sample selected from the group consisting of bone, mineralizingcartilage and tendon, dentin, cementum, the shells of mollusks, andother mineralized tissue of vertebrate, invertebrate, plant, bacterialand other organisms. In some embodiments, the method of the presentinvention may include any of the embodiments described above whereinsaid decalcification agent is tetrasodium-EDTA. In some embodiments, themethod of the present invention may include any of the embodimentsdescribed above wherein the ratio of the volume of said decalcificationagent to the approximate weight of said hard tissue sample is from about10:1 to about 20:1.

In some embodiments, the method of the present invention may include anyof the embodiments described above wherein the mixture is stirred forfrom about 3 days to about 7 days. In some embodiments, the method ofthe present invention may include any of the embodiments described abovewherein the decalcification agent added is changed from every about 36to every about 48 hours.

In some embodiments, the method of the present invention may include anyof the embodiments described above further comprising the step of addinga ribonuclease inhibitor to the liquid decalcification medium to preventdegradation of the RNA in said hard tissue sample. In some embodiments,the method of the present invention may include any of the embodimentsdescribed above wherein said method of preparing further includes thesteps of: removing cells from the sectioned tissue sample of using alaser; and extracting the RNA from said cells and analyzing said RNA forgene expression.

In another aspect, the present invention is directed to a kit forpreparing hard tissue samples for gene expression analysis using themethod of any of the embodiments described above. In some embodiments,the kit of the present invention may include any of the embodimentsdescribed above, further comprising a first container containing an RNApreservation solution; a second container containing a decalcificationagent solution; and a written protocol for using said solutions toprepare a hard tissue sample for later gene expression analysis. In someembodiments, the kit of the present invention may include any of theembodiments described above, wherein said decalcification agent solutionfurther comprises tetrasodium-EDTA. In some embodiments, the kit of thepresent invention may include any of the embodiments described above,wherein said first container further comprises a ribonuclease inhibitorto prevent degradation of the RNA in the tissue sample.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures in which:

FIG. 1 is a scanned image of 1% formaldehyde denaturing agarose gel. Asize marker was run in the first lane followed by RNA isolated from ratbones ground to powders under liquid nitrogen. Day 0 (0) was the controland was not immersed in decalcification solution. Subsequent lanenumbers refer to days rat bones were being demineralized indecalcification solution. RNA was observed to be intact across all timepoints and 28S and 18S r RNA bands were observed.

FIG. 2 is a graph reporting the raw data control plate with 11 humanreference genes from Applied Biosystems comparing one normal human bonedecalcified according to at least one embodiment of the presentinvention and its undecalcified counterpart and one diseased state(“slip”) human bone sample decalcified according to at least oneembodiment of the present invention and its undecalcified counterpart.The data show good correlation between sample halves.

FIG. 3 is a graph reporting the raw data shown in FIG. 2 normalized forthe number of cells per sample utilizing 18S rRNA. Little to novariation was noted in gene expression between 2 identical samples (onecontrol tissue bisected) and 2 identical samples (one disease-statetissue bisected) from RNA isolated before and after decalcificationaccording to at least one embodiment of the present invention. A lessthan 1 Ct difference is considered not statistically significant.

FIG. 4 is a photograph of a tissue sample from a young human proximalfemur decalcified according to at least one embodiment of the presentinvention and embedded in cryo-medium in preparation for beingsectioned. The tissue appears intact and complete with red marrowelements, white ribbon of the cartilage growth plate through the centerof the bone and the white outer layer which comprises the perichondrium.

FIG. 5A is an image taken at 4× magnification of sections cut from thehuman femur shown in FIG. 4 above that has been decalcified according toat least one embodiment of the present invention, cryo-sectioned,stained with eosin and prepared for laser capture microdissection(“LCM”) in this example. FIG. 5A was taken before LCM to show the intactsection.

FIG. 5B is an image taken at 4× magnification of the stained human femursection shown in FIG. 5A after LCM. The empty area where a group ofcells was removed by LCM is designated by a black star.

FIG. 6 is a graph reporting the results from the study of growth platecartilage in normal and SCFE patients shown above in FIGS. 4 and 5.These data showed the statistically significant downregulation ofextracellular matrix genes in the human SCFE condition.

FIG. 7 is a chart showing a microarray analysis of human biopsy samplesof control bones compared to SCFE bones utilizing frozen-ground samples.These results confirmed the data presented in FIG. 6 because thegrayed-in circles indicate statistical significance in skeletaldevelopment where the genes for type II collagen and aggrecan aregrouped.

FIG. 8 is an image of a mouse tibia decalcified according to at leastone embodiment of the present invention, cryo-sectioned and stained inpreparation for laser microdissection. The cell groups identified to beremoved by laser cutting are delineated by dashed lines and pointed outby black arrows.

FIG. 9 is a graph reporting the quantitative data generated frommicrodissection of the specific cell groups in the mouse tibia shownabove in FIG. 8.

FIG. 10 is graph reporting the quantitative data generated frommicrodissection of pig normal and hypothyroid proximal femur growthplates after being decalcified according to at least one embodiment ofthe present invention.

FIG. 11A is an image taken at 4× magnification of a representativesection cut from a pig proximal tibia that has been decalcifiedaccording to at least one embodiment of the present invention,cryo-sectioned onto PEN membrane slides, stained with eosin and preparedfor laser capture microdissection (“LCM”) in this example. FIG. 11A wastaken before LCM to show the intact section.

FIG. 11B is an image taken at 4× magnification of the stained pigproximal tibia section shown in FIG. 11A after LCM. The empty area iswhere a group of cells was removed by LCM.

FIGS. 11C and 11D are images taken at 4× of captured growth platechondrocytes from pig proximal tibia sections. From these cells, RNAwill be isolated and used in RT-qPCR analyses.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention is directed to a method of decalcification thatallows for the reproducible quantitative analysis of gene expression inhard tissue samples like bone, mineralizing cartilage and tendon,dentin, cementum, enamel and/or other tissues or organisms that are toohard to section effectively using conventional means.

In some embodiments of the method the present invention, a tissue samplecontaining a hard tissue is taken from a human, animal or plant subjectbeing studied. The human or animal tissue sample may be obtainedsurgically by any conventional means including by biopsy or by removalafter death. As used herein, the term “hard tissue” refers to anymineralized tissue including, but not limited to, bone, mineralizingcartilage and tendon, dentin, cementum, enamel, the shells of mollusks,and any other tissue of vertebrate, invertebrate, plant, bacterialand/or other organisms that is sufficiently mineralized to be difficultor impossible to cut into useful sections for analysis usingconventional means, including but not limited to, a cryostat. The tissuesample should be thin enough in at least one dimension to permit thedecalcification agent solution (discussed below) to fully infiltrate thesample. As will be appreciated by those of ordinary skill in the art,the size and shape of sample which can be decalcified will also dependon the type of sample, its location and function. Some samples containmore mineral and may be more or less compact (cancellous and corticalbone for instance). In some embodiments, the tissue sample may be asample of bone less than 5 mm, on any one side. In some embodiments, thetissue sample may be cut down to a size that will permit thedecalcification agent solution to fully infiltrate the sample.

Being a single-stranded nucleic acid, RNA is relatively fragile comparedto the double-stranded deoxyribonucleic acids (DNA) and has a relativelytransitory and/or temporary existence in the cell. Moreover, it is knownthat upon cell death, enzymes are released to begin breaking down RNA.So, while it is not required to practice the present invention, it isstrongly suggested that the tissue sample be placed in an RNApreservation solution as soon as possible after it is taken in order toprevent, or at least delay, the breakdown of the RNA in the sample.Suitable RNA preservation solutions are commercially available andinclude, for example, RNAlater® Solution made by Ambion, Inc.(Carlsblad, Calif.), but may be any medium that prevents or delaysdegradation of RNA. The RNA preservation solution should be usedaccording to its instructions with RNase-free conditions for the tissuesample being harvested. The amount of RNA preservation solution usedwill, of course, depend upon the particular solution chosen, but in someembodiments the volume of RNA preservation solution may be about 10times the volume-to-weight of the tissue sample. In some embodiments,tissue samples may be placed in RNAlater® Solution at 4° C. after beingtaken from the subject. In some embodiments, tissue samples may beimmediately frozen after being taken from the subject. In someembodiments, tissue samples may be taken from bones, teeth, joints,tendons, ligaments or other examples.

The tissue sample can be kept in the RNA preservation solution for anyperiod of time consistent with the written instructions and protocolsfor the RNA preservation solution provided by the manufacturer. In someembodiments, the tissue sample is placed in the RNA preservationsolution for a period from about 18 hours to about 24 hours at atemperature from about 3° C. to about 4° C., prior to decalcification.In some embodiments, the tissue sample is placed in the RNA preservationsolution for about 1 day at a temperature of about 4° C.

The decalcification solution may be prepared by adding a decalcificationagent to an RNA preservation solution. The decalcification agent may bea chelating agent. The decalcification agent must be capable of reactingwith and removing minerals, primarily calcium, found in the tissuesample without significantly damaging the surrounding tissues and mustdo so relatively quickly in order to limit the degradation of the RNA inthe sample. Suitable decalcification agents include, but are not limitedto, tetrasodium-ethylenediaminetetra-acetic acid (tetrasodium-EDTA) andtrisodium-ethylenediaminetetra-acetic acid (trisodium-EDTA), and saltsthereof. In some embodiments, the decalcification agent may betetrasodium-EDTA. The concentration of decalcification agent in thedecalcification solution will depend, of course, on the particularchelating agent being used but is ordinarily from about 9 to about 10grams of decalcification agent per every 90 ml of RNA preservationsolution. In some embodiments, the ratio of tetrasodium-EDTA salt (g) toRNA preservation solution (ml) is about 1:10.

The decalcification agent should also be either soluble in the RNApreservation solution or, to the extent not soluble, capable of forminga substantially stable and homogeneous mixture with the RNA preservationsolution. In some embodiments, the pH of the RNA preservation solutionand chelating agent mixture is increased to a pH of from about 8 toabout 10 and stirred for from about 15 minutes to about 30 minutes untilthe decalcification agent is fully dissolved in the RNA preservationsolution. In some embodiments, the pH of the RNA preservation solutionand chelating agent mixture/solution is adjusted by the dropwiseaddition of a base. In some embodiments, the pH of the RNA preservationsolution and decalcification agent mixture/solution is adjusted to a pHof from about 8 to about 9 to permit the decalcification agent todissolve. In some embodiments, the RNA preservation solution anddecalcification agent mixture/solution are stirred for from about 30minutes to about 60 minutes to permit the decalcification agent todissolve. In some embodiments, the RNA preservation solution anddecalcification agent mixture/solution are stirred for approximately 30minutes to permit the decalcification agent to dissolve.

Once the decalcification agent has dissolved in the RNA preservationsolution, the pH of the solution is then readjusted to be a pH ofbetween from about 7.2 to about 7.7, by any suitable means. In someembodiments, the pH of the solution is readjusted to be a pH of betweenfrom about 7.2 to about 7.4. In some embodiments, the pH of the solutionis readjusted to be a pH of about 7.4. In some embodiments, the pH ofthe solution is readjusted to be a pH of about 7.6. In some embodiments,the pH of the solution is readjusted by the dropwise addition of 6N HCl.

As will be appreciated by those of skill in the art, the RNA in humancells and more vascularized animal and plant cells is particularlysensitive and prone to degradation. When working with these types ofcells, a ribonuclease inhibitor may be added to the decalcificationsolution to further prevent, or at least limit, degradation of the RNAin these more sensitive samples. Many suitable ribonuclease inhibitorsare commercially available and include, but are not limited toSUPERase•In™ (Ambion, Inc., Carlsblad, Calif.). In some embodiments, theconcentration of ribonuclease inhibitor in the decalcification solutionmay be from about 30 U/ml to about 60 U/ml. In some embodiments, theconcentration of the ribonuclease inhibitor in the decalcificationsolution is about 50 U/ml.

As will be appreciated by those of skill in the art, the decalcificationsolution must be sterilized before it can be used. Sterilization may beperformed by any suitable method known in the art, but it is preferablydone by filtration through a filter having a pore size fine enough toremove bacteria and other microbes and into a suitable sterilecontainer. In some embodiments, the filter has a pore size of from about0.4 μm to remove large particles and then less than 0.2 μm forsterilization. In some embodiments, the decalcification solution issterilized by passing it through a 0.2 μm filter and into a suitablesterile container.

To begin decalcification, the tissue sample is immersed in an excess ofdecalcification solution prepared as set forth above. In someembodiments, the ratio of the volume of the decalcification solution tothe approximate weight of the hard tissue sample is from about 10:1 toabout 20:1. In some embodiments, the ratio of the volume of thedecalcification solution to the approximate weight of the hard tissuesample is about 20:1.

The tissue sample is kept in the decalcification solution for from about3 days to about 8 days depending upon the concentration of thedecalcification agent and the size and relative mineral content of thesample, until the sample has softened to the point that it can besectioned by conventional techniques. In some embodiments, thedecalcification solution containing the tissue sample may be stirred. Insome embodiments, the decalcification solution may be stirred for fromabout 3 days to about 7 days. In some embodiments, the sample is placedin the decalcification agent solution for 5 days.

The decalcification/chelating agent in the decalcification solutionchemically reacts with the mineral deposits, binding primarily calcium,reducing the crystal size and thereby solubilizing the mineral. Therecan be other metals in bone and other hard tissue but the main purposefor a chelating agent like tetrasodium-EDTA in this application is toremove the calcium from the hydroxyapatite, thereby leaving phosphateions in solution which have no calcium with which to bind and will bindwith the available sodium in the tetrasodium-EDTA.

The solubilizing of the hydroxyapatite calcium phosphate crystalsweakens or softens the hard tissue. It is believed that chelating agentslike tetrasodium-EDTA work by capturing metallic ions like calcium fromthe external layer of the apatite crystals within their structure,removing them from the hydroxyapatite crystals. When all of the calciumions from the outer layer of hydroxyapatite crystals have been captured,they are replaced by calcium ions from deeper layers. In this way, thecrystal size decreases gradually, producing an excellent gradualdecalcification of tissue components. This gradual decalcificationproduces an excellent means of maintaining tissue structure duringdecalcification and thereby excellent preservation of tissue components.

It also should be appreciated, however, that after some time thereaction will reach equilibrium and it may be necessary to periodicallychange the decalcification solution. In some embodiments, thedecalcification solution may be changed at intervals of from about 36hours to about 48 hours. In some embodiments, the decalcificationsolution is changed every 48 hours.

As set forth above, the sample should be kept in the decalcificationsolution until it becomes softened to the point that it can be sectionedby conventional techniques. However, it should be appreciated that thetissue sample need not be decalcified beyond the point necessary to workwith and/or section it. While it is believed that tetrasodium-EDTA andtrisodium-EDTA are better than the decalcification agents in the priorart, extended exposure to these chelating agents will gradually begin todegrade the RNA in the sample, and to the extent possible should beavoided. Extensive over-decalcification can destroy nucleic acids andproteins and results in poor sections where the tissue falls apart uponcutting with loss of morphological characteristics necessary foranalysis.

The softened tissue sample is then rinsed first in RNA preservationsolution and then in RNase-free or DEPC-treated water at a temperatureof from about 3° C. to about 4° C. The softened tissue sample is thenimmersed in a small beaker containing a commercially availablecryo-embedding medium for infiltration into the porous tissue that willresult in more intact sections. Suitable commercially availablecryo-embedding medium are known to those of skill in the art and mayinclude, but are not limited to, Cryo-OTC (Andwin Scientific,Schaumburg, Ill.) and Tissue Freezing Media (TFM™) (Triangle BiomedicalSciences, Inc., Durham, N.C.). Typically, 45-60 min at a temperature offrom about 3° C. to about 4° C. will be necessary before the sample issnap-frozen at minus 20° C. in a cryostat in preparation for sectioning.

Methods for analyzing specific cell types in heterogeneous tissuesamples have long been pursued with the microdissection of certain cellpopulations accomplished in the past by a skilled scientist and ascalpel. The technology of the present time utilizes automatedinstruments with lasers that are capable of dissecting out specificcells, cell types or populations of cells (such as diseased fromhealthy) from a tissue for downstream genomic and proteomic analyses.Laser microdissection requires the tissue to be sectioned and most oftenstained for viewing and identifying populations of cells of interest tobe microdissected and analyzed. Because this methodology can only beutilized on sectioned material, this invention allows for the analysisof such hard tissues that cannot be cut without decalcification.Decalcification can be accomplished by other products for suchapplications that involve protein and possibly DNA analysis but there isnot a methodology and solution available for consistent, reliable geneexpression (RNA) analysis. Laser microdissection provides theinvestigator with quantitative differences in downstream reversetranscription quantitative polymerase chain reaction (RT-qPCR) analyses.

This invention can also be utilized and is extremely beneficial forstudies involving in situ hybridization. Similar to lasermicrodissection, in situ hybridization requires sections of the tissueof interest to visualize the location of cell types in a heterogeneoussample. Unlike microdissection, cells are not removed from the tissue,but the RNA or gene of interest is identified by binding a labeledchromogenic or fluorescent probe to the section. In this way, RNAexpression can be microscopically identified and determined for specificareas or cell types. Although not a quantitative methodology per se,localization of the gene expression of molecules of interest can providea spatial map of events in the tissue. This methodology has been used inconjunction with laser microdissection in gene expression analysisstudies. This invention provides tissue sections of hard material thatare decalcified to retain the RNA intact for in situ probes to bind.

It has, moreover, been found that the decalcification process of thepresent invention provides RNA comparable in quality and quantity tothat provided by the snap-freezing the sample in liquid nitrogen andgrinding it to a powder, which is considered the gold standard in theart for RNA recovery from hard tissues. But unlike that method, thepresent method also makes it possible to determine the location in thesample from which the RNA was recovered.

In another aspect, the present invention is directed to a kit for use inthe preparation of hard tissues according to the methods describedabove. It is envisioned that the kit will comprise a container holdingan appropriate volume of RNA preservation solution, a container holdingan appropriate volume of decalcification solution as described above,and a written protocol for using the solutions to prepare a hard tissuesample for later gene expression analysis according to the method setforth above. In some embodiments, the decalcification solution maycontain tetrasodium-EDTA and the chelating and decalcification agent.

In light of the foregoing, it should be appreciated that the presentinvention significantly advances the art by providing a method ofdecalcification that allows the reproducible quantitative analysis ofgene expression for hard tissue samples that is an improvement over theprior art methods in a number of ways. While particular embodiments ofthe invention have been disclosed in detail herein, it should beappreciated that the invention is not limited thereto or therebyinasmuch as variations on the invention herein will be readilyappreciated by those of ordinary skill in the art. The scope of theinvention shall be appreciated from the claims that follow.

EXAMPLES

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.Further, while some of the examples may include conclusions about theway the invention may function, the inventors do not intend to be boundby those conclusions, but put them forth only as possible explanations.Moreover, unless noted by use of past tense, presentation of an exampledoes not imply that an experiment or procedure was, or was not,conducted, or that results were, or were not actually obtained. Effortshave been made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature), but some experimental errors and deviations maybe present. Unless indicated otherwise, parts are parts by weight,molecular weight is average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Synthesis of a Standard 50 ml Decalcification SolutionAccording to One Embodiment of the Present Invention

40 ml of RNAlater® was measured into a 100 ml baked beaker (RNase-free)at room temperature. 5 g tetrasodium-EDTA (ethylenediaminetetra-aceticacid) salt was then added as a decalcification agent. The solution pHwas then adjusted to be above 8.0 by drop wise addition of 10% NaOH. Thesolution was stirred for about 30 minutes, until all of thetetrasodium-EDTA salt had dissolved. The solution volume was thenadjusted to 50 ml with additional RNAlater® and the pH readjusted to 7.6by dropwise addition of 6N HCl. The total solution was then sterilizedby passing it through a 0.2 μm filter into a sterile conical tube.

Example 2 Analysis of RNA Integrity after Decalcification of Mouse BoneSamples for Sectioning within a Short Time Frame

First, 5 G of ethylenediamine tetraacetic acid as a tetrasodium salt wasplaced in 40 ml of RNAlater® Solution (Ambion, Life Technologies). Whenthe tetrasodium-EDTA did not initially dissolve, the pH was increased toabove 8.0 by the addition of sodium hydroxide (10N) with stirring for30-60 min. After dissolution, the pH was decreased to 7.6 with acid (6NHCL). The solution volume was adjusted to a final volume of 50 ml withRNAlater® Solution. The homogenous mixture was sterile filtered througha 0.2 μm filter in preparation for use. This solution was used in aninitial experiment to determine time frame of the process ofdecalcification in relation to RNA degradation and feasibility of itsuse at 10% tetrasodium-EDTA concentration.

Second, sixteen mouse tibial bones (left and right rear sides), 1.6-1.9cm in length and less than 5 mm in diameter, were harvested from eightblack mice (CD-1) that were 8-10 months old. Two bones were flash frozenin liquid nitrogen and stored at minus 80° C. (day 0). The remaining 14bones were placed in RNAlater® Solution for approximately 24 hrs at 4°C. After 24 hrs, the bones were divided into two beakers (right and leftsides) containing 10 ml of decalcification solution made as set forthabove. Mouse tibial bones weigh 75-100 mg each and approximately 700 mgor 7 bones is equivalent to 7 ml of decalcification solution (10×).Decalcification solution was changed on days 2, 3, 6 and 8.

Third, two mouse bones, one from left side beaker and one from rightside beaker, were removed from the decalcification solution at 7 timepoints, 24 hrs, 2, 3, 6, 7, 8, and 9 days. Upon removal, they were flashfrozen in liquid nitrogen and stored at minus 80° C. All samples wereanalyzed for RNA quality and quantity and compared to day 0.

The mouse bones began to soften by day 3 and by day 9 were malleable.RNA quality and quantity decreased by day 9 according to UV 260/280ratios.

Fourth, four additional mice tibias were harvested and placed inRNAlater® Solution at 8:30 am at 4° C. Five grams of tetrasodium-EDTAwas added to a 100 ml beaker containing 40 ml of RNAlater® Solution. pHwas adjusted to 9 with 10N NaOH and stirred for 45 min to dissolve thetetrasodium-EDTA. pH was reduced to 7.6 with acid and volume adjusted to50 ml with RNAlater® Solution. At 5 μm, 20 ml of cold decalcificationsolution was added to the 4 mouse bones and stirred. After 40 hrs, thefirst mouse tibia was removed and allowed to rinse in RNAlater® Solutionfor 8 hrs. The bone was embedded in tissue freezing media in a cryostat(−20° C.) and sectioning was attempted but the bone was still toodifficult to cut. Another tibia was removed at 48 hrs and placed inRNAlater® Solution to rinse overnight and the decalcification solution(15 ml) was changed. The final two bones were subsequently removed fromdecalcification solution at 72 and 96 hrs with decalcification solution(15 ml) changed again after 72 hrs. The final solution at 96 hrs wastested with ammonium oxalate for completeness of demineralization andfound to be complete.

Last, the remaining three decalcified bones from subsequent days wereembedded in tissue freezing in a cryostat (−20° C.) and sectioning wasaccomplished without difficulty. This example showed small compact bone(1.6-1.9 cm by 3-4 mm) could be sectioned after 48 hrs withoutdifficulty with this invention and over decalcification could lead toloss of RNA quantity and quality.

Example 3 Analysis of RNA Integrity after Decalcification of Rat BoneSamples for Sectioning within a Short Time Frame

First, 10.0 grams of tetrasodium-EDTA was weighed out into a 150 mlsterile beaker and 80 ml of RNAlater® Solution was added. The pH of themixture was adjusted to 8.8 and the tetrasodium-EDTA was dissolved withstirring. The pH was then reduced to 7.6 and RNAlater® Solution wasadded to an adjusted final volume to 100 ml. The solution was sterilizedby passing it through a 0.2 μm filter and into a sterile container forsubsequent use. The solution was stored at 4° C.

Second, the right and left tibias were removed from 5 adult rats. Thetibias weighed approximately 1 gram and were 3 cm long by 0.5 cm indiameter. All 10 tibias were placed in RNAlater® Solution forapproximately 24 hrs at 4° C. After 24 hrs, one tibia was removed andflash frozen in liquid nitrogen and stored at minus 80° C. (day 0).Remaining 9 bones were placed in 100 ml of decalcification solution withslow, constant stirring at 4° C.

Third, one tibia each was removed after decalcification for 1, 2, 3, 4,5, 6, and 7 days with two remaining bones removed after day 8. Each bonewas immediately flash frozen in liquid nitrogen upon removal and storedat minus 80° C. Decalcification solution was only changed on days 3 and6. On day 3, the bone would not indent with pressure from a blunt metalpoint of a forceps. On day 4, 10 ml of decalcification solution wasremoved and checked for completeness of demineralization with 5%ammonium oxaloacetate but a precipitate formed indicating the bones wereonly partial decalcified. By day 5, the rat bone was indenting orsoftening when touched with the forceps but just close to cartilaginousendplates. On day 6, the thick cortical midshaft was still hard withlittle deformation from forceps. By day 8, remaining rat bones wereremoved since there appeared to be no change from previous 2 days.

Last, RNA was extracted from all collected bones ground to powder andanalyzed by a 1% formaldehyde agarose gel. See FIG. 1. A size marker wasrun in the first lane followed by RNA isolated from rat bones ground topowders under liquid nitrogen (gold standard) on subsequent daysstarting with day 0. Days indicate the amount of time the bone wasimmersed in decalcification solution. RNA was intact across all timepoints by 28S and 18S rRNA bands observed in all lanes. FIG. 1. It wasconcluded that mouse and even thicker rat bones can be decalcified usingthe method set forth above in a time frame where they can be sectionedand RNA quality is maintained.

Example 4 Quantitative Analysis of Gene Expression of Bones Bisectedwhere Half are not Decalcified and the Other Half Decalcified in NewDecalcification Solution

First, 10.0 grams of tetrasodium-EDTA was weighed out into a 150 mlsterile beaker and 80 ml of RNAlater® Solution was added. The pH of themixture was adjusted to 9 and the tetrasodium-EDTA was dissolved withstirring. The pH was then reduced to 7.6 and RNAlater® Solution wasadded to an adjusted final volume to 100 ml. The solution was sterilizedby passing it through a 0.2 μm filter and into a sterile container forsubsequent use. The solution was stored at 4° C.

Second, eight mouse tibia bones (1.4-1.6 cm by 3-4 mm diameter) wereharvested from four black mice (CD-1) that were 3-4 months old. The fourleft tibias and four right tibias were placed in 10 ml each of RNAlater®Solution for approximately 24 hrs at 4° C. After 24 hrs, the bones weredivided into half with bone scissors and placed in two beakerscontaining 10 ml each of decalcification solution prepared as set forthabove.

Third, eight tibial halves were ground to powder under liquid nitrogen(the “gold standard” for RNA recovery) while the other correspondinghalves were decalcified in the decalcification solution prepared above.After 24 hrs, the four left tibias were removed and rinsed in RNAlater®Solution for 1 hr and then these were also ground to a powder. Thedecalcification solution was then changed and after an additional 24 hrs(48 hrs total), the remaining four right tibias were removed, rinsed inRNAlater® Solution for 1 hr and ground to a powder.

This experimental methodology allowed for a more robust paired-samplet-test statistical analysis (Sig. 2-tailed) of genes common to theextracellular matrix of hard tissues. The analysis results are shown inTable 1. Matrix genes, osteopontin (OPN), osteocalcin (OC), type Icollagen (Type I), aggrecan (Agg), bone sialoprotein (BSP) and areference gene cyclophilin D (Cyc) were examined. No statisticalsignificance (p>0.05) was found between the undecalcified (“Un”) samples(RNA removed using “gold standard” freezing and powdering technique) andsamples (“Decal”) utilizing the decalcification method of the presentinvention.

TABLE 1 Paired Samples Test Degrees of Freedom (Df) Sig. (2-tailed) Pair1 OPN Decal - OPN Un 8 .140 Pair2 Cyc Decal - Cyc Un 7 .090 Pair 3 OCDecal - OC Un 8 .167 Pair4 TypeI Decal - TypeI Un 8 .126 Pair 5 AggrecnDe - Agg Undec 6 .606 Pair6 BSP Decal - BSP Undec 8 .385

Example 5 Decalcification of Human Core Biopsies from Human ProximalFemur, Distal Femur, Proximal Tibia and Fibula

First, 10.0 grams of tetrasodium-EDTA was weighed out into a 150 mlsterile beaker and 80 ml of RNAlater® Solution was added. The pH of themixture was adjusted to 8-9 and the tetrasodium-EDTA was dissolved withstirring after approximately 30 minutes. The pH was then reduced to 7.6with a weak acid and RNAlater® Solution was added to an adjusted finalvolume to 100 ml. The solution was sterilized by passing it through a0.2 μm filter and into a sterile container. The solution was stored at3-4° C. for subsequent use.

Second, core biopsies were obtained surgically from patients who hadslipped capital femoral epiphysis (SCFE) and leg length discrepancies.These samples, ranging in diameter from 2-5 mm and length of 1-3 cm,were preserved in RNAlater® Solution upon removal from the patients.After 24 hrs in RNAlater® Solution at 4° C., they were decalcified insterile solution prepared above for 5 days. Initially, one control(“ctrl”) sample and one disease-state sample (“slip”) were cut in halfwith a dremmel saw. One half was not placed in the above-describeddecalcification solution, but instead was ground to a powder in a Spexgrinder mill under liquid nitrogen. The other half was placed in theabove-described decalcification solution until the hard tissue softenedand could be cut with a cryostat knife. Total RNA was isolated accordingto the TriReagent protocol and reverse transcribed to cDNA. The cDNA wasutilized in Experiment 1, below.

Experiment 1

A human endogenous control plate was purchased from Applied Biosystemsand a comparison was made between the bisected samples (“ctrl” and“slip”) decalcified (“decal”) and undecalcified (“unde”) halves. Theseresults are represented in FIGS. 2 and 3. FIG. 2 shows the raw datawhere lane 1 is an internal control and lane 2 represents 18S rRNA whichis equivalent to the amount of cDNA loaded into each well. There is agood correlation between the gene expression in the decalcified controlsample and its undecalcified counterpart. Also, there were only minordifferences in gene expression when the SCFE decalcified sample wascompared to its undecalcified counterpart. According to AppliedBiosystems protocols, a 1 Ct difference between samples is notconsidered statistically significant. FIG. 3 normalizes the gene data to18S rRNA and thus obviates the loading differences or cell numbersbetween the decalcified and undecalcified counterparts. These datademonstrate a strong correlation for gene expression between theundecalcified sample half and the decalcified half.

Experiment 2

Other human femurs, tibias and fibulas removed during routine orthopedicprocedures were placed in RNAlater® Solution immediately followingsurgery. Any sample with their length, width and depth dimensions allgreater than 5 mm were bisected or reduced by the surgeon to have athickness of 5 mm for adequate solution infiltration. After 24 hrs inRNAlater® Solution at 4° C., bone samples were immersed in cold (4° C.)decalcification solution (item 1) in a 10-20× volume to weight. Thedecalcification solution was changed every approximately 48 hrs andsamples were removed when they were judged to be softening from pressureof a forceps. Smaller bones, typically core samples from the tibias andfibulas (0.5-1×0.8-1×0.8-1 cm), were decalcified for 3 days and thelarger femurs (0.5-1×1−1.5×1.2-2 cm) were removed after 5 days in theabove-described decalcification solution.

All decalcified samples were in RNAlater® Solution for approximately 24hrs at 4° C. to remove decalcification solution. RNAlater® Solution wasbriefly washed off with cold RNase-free water for 30 sec and sample wasplaced in cold tissue embedding medium to prepare for sectioning. Thesample morphology appeared preserved with the bone and marrow elementsvisible and the white cartilage components of the growth plate andperichondrium evident in the example of a proximal femur shown in FIG.4. The samples were sectioned (4-5 μm) onto clean glass slides, fixedand stained with eosin for laser capture microdissection (FIG. 5A).After LCM (FIG. 5B), cells were lysed, total RNA was isolated andreverse transcribed for qPCR. Results of this study are reported ingraphic form in FIG. 6. The downregulation of the extracellular matrixgenes of SCFE patients in this study was subsequently confirmed by amicroarray analysis utilizing RNA isolated by the standard grinding ofcontrol and SCFE samples under liquid nitrogen (FIG. 7). Thedownregulation of extracellular matrix genes is noted in the grayed areathat is labeled as skeletal development. FIG. 7.

Example 6 Mouse Study of Tibial Development with Exercise

First, 10.0 grams of tetrasodium-EDTA was weighed out into a 150 mlsterile beaker and 80 ml of RNAlater® Solution was added. The pH of themixture was adjusted to 9 with a base and the tetrasodium-EDTA wasdissolved with stirring after approximately 30 minutes. The pH was thenreduced to 7.6 with an acid and RNAlater® Solution was added to anadjusted final volume to 100 ml. The solution was sterilized by passingit through a 0.2 μm filter and into a sterile container. The solutionwas stored at 3-4° C. for subsequent use.

Eight mice were randomly divided into either cage-control or exercisegroups. The exercise group of mice (n=4) were run on a treadmill forseveral hours per day. At the end of the experiment, the mice weresacrificed and their tibias harvested and preserved in RNAlater®Solution at 4° C. After 24 hours, tibia bones measuring 1-2 mm by 1.5-2cm were placed in 15 ml (10-20×) of decalcification solution withstirring for 5 days. The decalcification agent solution was changedevery 36-48 hrs. After 5 days of decalcification, the tibia bones wererinsed in RNAlater® Solution for approximately 24 hrs at 4° C. TheRNAlater® Solution was briefly washed off with cold RNase-free water for30-60 sec and the sample was placed in cold tissue embedding media toprepare it for sectioning. Each control (n=4) and exercised mouse tibia(n=4) was sectioned 4-5 μm thick in a cryostat with a tungsten carbideblade. Each bone was completely sectioned from the anterior to posteriorface comprising over 100 sections per mouse. Two areas of all tibialsections comprising the articular surface (AS) and growth plate (GP)were laser captured and a representative section is illustrated in FIG.8. The lighter stained areas that are outlined by dashed lines andarrows were the groups of cells to be removed by LCM. These groups ofcells, representing two different areas of the mouse bone, were analyzedby isolation of their RNA. Downstream gene expression analysis revealedquality RNA by reference genes 18S rRNA and elongation factor 1(involved in transcription). A statistical analysis was preformed todetect extracellular matrix gene differences related to exercise. Thedata is reported in graphic form in FIG. 9.

Example 7 Rabbit Study of Compression on Growth Plate Development

Pins were surgically implanted across the top and bottom of the rightand left proximal tibial growth plates of 12 week-old New Zealand whiterabbits. On one leg, a compressive device was added to the pins thatapplied forces of either 10N (n=8) or 30N (n=8) (experimental samples)for either 2- or 6-weeks. The contralateral leg served as a sham control(n=16). The rabbits were sacrificed at the appropriate time point andtibial bones were harvested, bisected and placed in RNAlater® Solutionfor approximately 24 hrs. Gene studies in this experiment wereaccomplished by grinding bisected halves under liquid nitrogen to apowder and then isolating the RNA.

20.0 grams of tetrasodium-EDTA was weighed out into a 250 ml sterilebeaker and 160 ml of RNAlater® Solution was added. The pH of the mixturewas adjusted to 9.5 with a basic solution (NaOH) and thetetrasodium-EDTA was dissolved with stirring after approximately 45minutes. The pH was then reduced to 7.6 with an acid and RNAlater®Solution was added to an adjusted final volume to 200 ml. The solutionwas sterilized by passing it through a 0.2 μm filter and into a sterilecontainer. The solution was stored at 3-4° C. for subsequent use.

One bisected sample (approx. 0.5×1×2 cm) from each group (2 wk controland experimentals and 6 wk control and experimentals) was decalcifiedseparately (n=6 bones) using a decalcification agent solution of 15 mleach. The solution was changed once on day 2 and softened bones wereremoved after 4 days and rinsed in RNAlater® Solution to removedecalcification solution for approximately 24 hrs at 4° C. The RNAlater®Solution was briefly washed off with cold RNase-free water for 30-60 secand the sample was placed in cold tissue embedding media to prepare forsectioning. Each control (n=2) and experimental rabbit bone (n=4) wassectioned 4-5 μm thick in a cryostat with a tungsten carbide blade.Growth plate chondrocytes from tibial bones of the rabbits were lasercaptured by microdissection and gene expression analysis was comparedthe same analysis from bones that were ground to a powder.

Example 8 Pig Study of Hypothyroidism on Proximal Femur Development

Four miniature swine were divided into two groups and one group (n=2)was given a drug to decrease their thyroid function. After 14 weeks, thepigs were sacrificed and the proximal and distal femurs and the proximaltibias were harvested. Surgeons bisected the pig bones and then placedthem in RNAlater®Solution for approximately 24 hrs at 4° C. After oneday, the samples were snap-frozen and stored at minus 80° C. untilanalysis.

20.0 grams of tetrasodium-EDTA was weighed out into a 250 ml sterilebeaker and 160 ml of RNAlater® Solution was added. The pH of the mixturewas adjusted to 9.5 with a basic solution (NaOH) and thetetrasodium-EDTA was dissolved with stirring after approximately 45minutes. The pH was then reduced to 7.6 with an acid and RNAlater®Solution was added to an adjusted final volume to 200 ml. The solutionwas sterilized by passing it through a 0.2 μm filter and into a sterilecontainer. The solution was stored at 3-4° C. for subsequent use.

The pig bones used had varying dimensions of 0.3-0.5×1-2.5×2-3.0 cm.First, the four proximal femurs (size approx. 0.5×2.5×1.7 cm) wereseparately decalcified in 20 ml each of decalcification solution afterremoval from minus 80° C. storage. The four proximal femurs weredemineralized for 5 days at 4° C. with the solution changed on days 2and 4. The softened pig proximal femurs were rinsed in RNAlater®Solution for approximately 24 hrs at 4° C. The RNAlater® Solution wasbriefly washed off with cold RNase-free water for 30-60 sec and thesample was placed in cold tissue embedding media to prepare forsectioning. Each control (n=2) and hypothyroid (n=2) bone was sectioned4-5 μm thick in a cryostat with a tungsten carbide blade. RNA wasisolated from laser captured growth plate cartilage and gene expressioncompared by RT-qPCR. Results were highly correlated between sample typesresulting in a statistical significance by student's t-test for n of 2in FIG. 10.

The following decalcification solution was used with the four proximaltibias and four distal femurs. 40.0 grams of tetrasodium-EDTA wasweighed out into a 500 ml sterile beaker and 350 ml of RNAlater®Solution was added. The pH of the mixture was adjusted to 9.5 with abasic solution (NaOH) and the tetrasodium-EDTA was dissolved withstirring after approximately 45 minutes. The pH was then reduced to 7.6with an acid and RNAlater® Solution was added to an adjusted finalvolume to 400 ml. The solution was sterilized by passing it through a0.2 μm filter and into a sterile container. The solution was stored at3-4° C. for subsequent use.

Subsequently, the four proximal tibias (approx. 0.5×0.9×1.4 cm) and fourdistal femurs (approx. 0.5×0.8×3 cm) were removed from minus 80° C.storage and placed in separate beakers containing 20 ml of colddecalcification solution for each bone. The solution was changed after48 hrs and the softened bones were removed after 4 days to RNAlater®Solution to remove decalcification solution for approximately 24 hrs at4° C.

The RNAlater® Solution was briefly washed off with cold RNase-free waterfor 30-60 sec and the sample was placed in cold tissue embedding mediato prepare for sectioning. Each control (n=2) and hypothyroid (n=2) bonewas sectioned 15-20 μm thick in a cryostat with a tungsten carbide bladeonto new plastic (PEN) membrane slides. These new slides althoughdifficult to use allow for more cells to be isolated in a shorter timeframe. This invention made it a possibility to use an antiroll platedevice in the cryosectioning art for section transfer to the PENmembrane slides. An example of the section on the slide is shown in FIG.11. FIG. 11A is the view under the laser capture microscope of thestained and prepared section. FIG. 11B is the section after removal ofcells and in FIGS. 11C and D are representative caps showing thecaptured isolated cell groups. RNA was then isolated from laser capturedgrowth plate cartilage of these pig bones and gene expressioncomparisons by RT-qPCR of control and hypothyroid animals is ongoing.

What is claimed is:
 1. A method of preparing a decalcification agentsolution for the decalcification of hard tissues like bone, mineralizingcartilage and tendon, dentin, cementum and enamel without degrading theribonucleic acids (RNA) contained therein, comprising the steps of: A.providing an RNA preservation solution; B. adding a decalcificationagent to said RNA preservation solution; wherein said decalcificationagent further comprises tetrasodium-ethylenediaminetetra-acetic acid(tetrasodium-EDTA) or trisodium-ethylenediaminetetra-acetic acid(trisodium-EDTA); C. adjusting the pH of the mixture of Step B to a pHof from about 8 to about 10 and stirring until the decalcification agentis substantially dissolved. D. adjusting the pH of the mixture of Step Cto a pH of from about 7.4 to about 7.7; and E. sterilizing andcollecting the mixture of Step D in a sterilized container.
 2. Themethod of claim 1 wherein said decalcification agent istetrasodium-ethylenediaminetetra-acetic acid (tetrasodium-EDTA).
 3. Themethod of claim 1 wherein the mixture of Step C is stirred for fromabout 30 minutes to about 60 minutes.
 4. The method of claim 1 whereinthe pH of the mixture of Step C is adjusted to a pH of about 7.6.
 5. Themethod of claim 1 wherein the mixture of Step E is sterilized byfiltration.
 6. The method of claim 5 wherein the mixture of Step D issterilized using a 0.2 μm filter.
 7. A method of decalcification of hardtissues like bone, mineralizing cartilage and tendon, dentin, cementumand enamel for sectioning without degrading the ribonucleic acids (RNA)contained therein, comprising the steps of: A. obtaining a hard tissuesample and placing it in an RNA preservation solution; B. removing saidhard tissue sample from the RNA preservation solution and placing it ina liquid decalcification medium comprising an RNA preservation solutionand a decalcification agent, wherein said decalcification agent isselected from the group consisting of tetrasodium-EDTA or trisodium-EDTAwherein said decalcification agent begins to remove calcium from mineraldeposits in said hard tissue sample; C. stirring the mixture of step Bsaid until said hard tissue sample softens for sectioning; and D.rinsing the softened hard tissue sample of step C in an RNA preservationsolution to remove any remaining decalcification agent.
 8. The method ofclaim 7 wherein said hard tissue sample is a tissue sample selected fromthe group consisting of bone, mineralizing cartilage and tendon, dentin,cementum and enamel.
 9. The method of claim 7 wherein the size of saidhard tissue sample does not exceed 0.5 cm, on one side.
 10. The methodof claim 7 wherein said hard tissue sample is placed in said RNApreservation solution for a period of from about 18 hours to about 24hours at a temperature of from about 3° C. to about 4° C.
 11. The methodof claim 7 wherein said hard tissue sample is placed in said RNApreservation solution for about 1 day at a temperature of about 4° C.12. The method of claim 7 wherein said liquid decalcification medium isthe decalcification agent solution of claim
 1. 13. The method of claim 7wherein said decalcification agent istetrasodium-ethylenediaminetetra-acetic acid (tetrasodium-EDTA).
 14. Themethod of claim 7 wherein the ratio of the volume of saiddecalcification agent to the weight of said hard tissue sample is fromabout 10:1 to about 20:1.
 15. The method of claim 7 wherein the mixtureof Step B is stirred for from about 3 days to about 7 days.
 16. Themethod of claim 7 wherein the liquid decalcification medium of step B ischanged from every about 36 to every about 48 hours.
 17. The method ofclaim 7 wherein the decalcification agent added in step B is removedfrom said tissue sample by rinsing said tissue sample in an RNApreservation solution for a period of time from about 12 hours to about24 hours.
 18. The method of claim 7 further comprising the step of: E.rinsing the tissue sample in RNase-free water to remove any remainingRNA preservation solution.
 19. A method of preparing hard tissues likebone, mineralizing cartilage and tendon, dentin, cementum and enamel forgene expression analysis comprising the steps of: A. obtaining a hardtissue sample; B. trimming said hard tissue sample to a minimum sizeneeded for gene expression analysis, wherein said tissue sample has atleast one edge that is less than 0.5 cm in length; C. placing said hardtissue sample in an RNA preservation solution; D. removing said hardtissue sample from the RNA preservation solution and placing it in aliquid decalcification medium comprising a decalcification agentselected from the group consisting of tetrasodium-EDTA or trisodium-EDTAand an RNA preservation solution, wherein said decalcification agentbegins to remove calcium from mineral deposits in said hard tissuesample, causing said hard tissue sample to soften; E. stirring themixture of step D until enough of the calcium has been removed from themineral deposits in said hard tissue sample to cause the tissue sampleto soften enough to permit sectioning of said tissue sample; F. rinsingthe softened hard tissue sample of step E in RNA preservation solutionand then RNase-free water; G. soaking the softened hard tissue sample ofstep F in cryoembedding medium; and H. freezing and cryosectioning thesoftened hard tissue sample of step G.
 20. The method of claim 19wherein said hard tissue sample is a tissue sample selected from thegroup consisting of bone, mineralizing cartilage and tendon, dentin,cementum and enamel.
 21. The method of claim 19 wherein said liquiddecalcification medium is the decalcification agent solution of claim 1.22. The method of claim 19 wherein said decalcification agent istetrasodium-EDTA.
 23. The method of claim 19 wherein the ratio of thevolume of said decalcification agent to the approximate weight of saidhard tissue sample is from about 10:1 to about 20:1.
 24. The method ofclaim 19 wherein the mixture of Step B is stirred for from about 3 daysto about 7 days.
 25. The method of claim 19 wherein the decalcificationagent added in step B is changed every from about 36 to about 48 hours.26. The method of claim 19 further comprising the step of adding aribonuclease inhibitor to the liquid decalcification medium of step C toprevent degradation of the RNA in said hard tissue sample.
 27. Themethod of claim 19 wherein said method of preparing further includes thesteps of: I. removing cells from the sectioned tissue sample of step Husing a laser; and J. extracting the RNA from said cells and analyzingsaid RNA for gene expression.
 28. A kit for preparing hard tissuesamples for gene expression analysis using the method of claim
 20. 29.The kit of claim 28 comprising a first container containing an RNApreservation solution; a second container containing a decalcificationagent solution; and a written protocol for using said solutions toprepare a hard tissue sample for later gene expression analysis.
 30. Thekit for gene expression analysis of claim 29 wherein saiddecalcification agent solution further comprises tetrasodium-EDTA. 31.The kit for gene expression analysis of claim 29 wherein said firstcontainer further comprises a ribonuclease inhibitor to preventdegradation of the RNA in the tissue sample.